Chromatographic methods are commonly used to separate a mixture into various components, so that these components can be identified and quantified. A variety of mechanisms are utilized to produce the desired separation between the components of the mixture. In one class of systems, the wall of the tube through which the mixture flows is coated with a material that exhibits different affinities for different components of the mixture. The speed of migration along the channel is greater for components exhibiting a weaker affinity for the wall, thereby producing a separation of mixture components according to their affinity for the coated wall. In another class of systems, the column is packed with a material, such as a gel or particulate, that provides the differential interaction with the components of the mixture.
These separation columns typically pass the sample solution past a detector that measures some physical property of the components, such as the light absorbance, the fluorescence emission, the refractive index or the electrical conductivity of the sample. In each of the first three of these particular cases, a light beam is passed through the sample. For the case of absorbance measurements in a capillary column, a detector is positioned adjacent to the capillary and opposite to a light source on an optical axis perpendicular to the capillary, such that the detector receives light after it passes through the sample. Fluorescence detection exhibits the advantages of superior selectivity and sensitivity in detection of many compounds.
Because absorbance and fluorescence signals are typically relatively weak, it is important to understand the fluorescent and non-fluorescent light distributions in order to maximize a performance parameter of the system, such as the signal-to-noise ratio ("S/N ratio"), the gain of the detected signal, some combination of these two signals (e.g., the minimum detectable concentration) or some other performance parameter appropriate for optimizing the system. The gain is defined to be equal to dS/dC, where S is the amplitude of the detector signal and C is the concentration of the sample. The minimum detectable concentration (MDC) is the minimum concentration that can be detected by the system and is defined to occur at that concentration for which the signal S is twice the noise signal of the system. The MDC is determined by plotting the signal S as a function of concentration and determining the concentration at which this curve has a signal value equal to twice the noise.
In the fluorescent systems presented in the article On-Column Capillary Flow Cell Utilizing Optical Waveguides For Chromatographic Applications by Alfredo E. Bruno, et al, Anal. Chem. 1989, 61, p. 876-883, optical fibers are utilized to carry incoherent light to the flowcell and are also utilized to transmit light to a detector. For a capillary of specified inner and outer diameters, a ray path calculation is presented that calculates the distribution of scattered light and the fraction of incident light that actually passes through the bore of the capillary. Because there is no focussing of the light from the light from the optical fiber onto the collecting optical fiber, the collection efficiency of the collecting optical fiber is limited by its acceptance angle and the spacing between the output end of the exposing optical fiber and the input end of this collecting optical fiber. Typically, each of these fluorescent systems collects less than one-eighth of the fluorescent light emitted from that system.
Because the use of small diameter bore capillaries improves the separation between the components of a sample, capillary liquid chromatography uses columns with very small internal diameters (typically 5 to 300 microns). The use of a small inner diameter capillary is also advantageous, because it reduces the amount of sample that is needed to fill the capillary during a measurement. Unfortunately, because the optical beam is typically directed substantially perpendicular to a central axis of the capillary, such reduced inner diameter also reduces the pathlength of the light through the sample, thereby degrading the signal-to-noise ratio of such measurements.
The S/N ratio has been improved by use of stronger light sources and/or coherent light sources in which the energy is concentrated at a wavelength that is particularly efficient at producing fluorescence. This improvement therefore arises from an increased amplitude of the signal. However, part of the noise component, such as scattered light, is proportional to the intensity of the exposing light. Therefore, when this portion of noise is a significant fraction of the total noise, it is important to minimize this noise component, because in such cases the signal-to-noise ratio cannot be significantly improved by use of a more intense beam of exposing light. This is particularly true for fluorescence measurements, because of the low signal level typically encountered in fluorescence measurements. It is therefore advantageous to increase the pathlength of the beam of light through the sample in order to increase the sensitivity of the system.
In the flow cell presented in U.S. Pat. No. 5,057,216, entitled Capillary Flow Cell, issued to Jean-Pierre Chervet on Oct. 14, 1991, the capillary is bent into the shape of a Z to enable a beam of exposing light to be directed along the central leg of this Z-shape, thereby increasing the pathlength from the inner diameter of the capillary to the length of this central leg of the Z-shaped capillary. A flowcell in which a portion of the capillary is bent into the shape of a Z will be referred to herein as a "Z-Cell". In a Z-Cell, the capillary has a first bend, referred to herein as the "entrance elbow" of the Z-Cell, through which some of the beam of exposing light can be passed into a straight section of the capillary and then out of a second bend, referred to herein as the "exit elbow" of the Z-Cell. The straight section of the capillary between these two elbows is referred to herein as the "central leg" of the Z-Cell. The protective coating is removed from this capillary in the elbow regions to enable this light to pass into and out of the capillary flow cell. The increased pathlength of the light through the sample should produce a corresponding increase in the sensitivity of this detection system.
Although this patent indicates that a sensitivity enhancement of at least 100 should result for this detection system in comparison to the traditional capillary detection system in which the incident light beam is perpendicular to the axis of the capillary, actual measurements of the sensitivity show that the sensitivity enhancement is closer to 4.5. Therefore, this approximately 20-fold shortfall in sensitivity from that which would be expected indicates that the exposing light is not concentrated into the bore of the capillary, but that, instead, effectively only about 5% of this beam passes through the bore in the central leg of the Z-Cell. This not only substantially offsets the sensitivity gain due to the increased pathlength, it also contributes to the noise component, because of the increased fraction of the incident beam that does not pass through the central leg can contribute to the noise component of the measured signal, but does not contribute to the signal component.
As discussed in the next section, this occurs because there does not appear to be any recognition of the effect of the entrance elbow on the incident beam of exposing light and the resulting detector response. In order to maximize the amount of light energy in the cell, at least one and preferably all of the following parameters of the beam be selected to maximize the product of the intensity and average pathlength of the exposing light through the central leg of the capillary: the diameter of the beam at the entrance elbow of the capillary; the direction of incidence of this beam on the entrance elbow relative to the axis of the central leg of the capillary; the amount of lateral offset of this beam, relative to the central leg of the capillary, at the point of incidence of the beam on the capillary; the degree of collimation of this beam at the entrance elbow; and the radius of curvature of the entrance elbow.